Epirubicin, olaparib and ribociclib are three anti-tumoral molecules currently used in cancer therapy. Since serum levels of these drugs reveal a large inter-individual variability, validation of a fast, simple and reliable analytical method for quantitative determination of these molecules in human serum, with Therapeutic Drug Monitoring (TDM) purposes would be necessary. Papers in literature describe separation, identification and quantitation of the drugs in pharmaceutical formulas and biological matrices. The large majority of these approaches use protein precipitation for sample pre-treatment, followed by LC separation and tandem-mass-spectrometry (MS/MS) detection. We propose an alternative method using Solid Phase Extraction (SPE) with Oasis PRiME HLB® cartridges followed by high-performance liquid chromatography (HPLC) using a C18 (4.6 × 50 mm) column. A gradient mobile phase with 0.1% formic acid/acetonitrile was utilized. MS detection in single ion recording (SIR) mode was employed. A 13-minutes run-time analysis, including column re-equilibration was assessed. Data for all molecules were validated according to ICH Guidelines. Calibration curves for all analytes were linear with correlation coefficient larger than 0.997. Values for precision were less than 6%. The method was applied on serum samples from patients up-taking the drugs, proving its suitability in TDM assisted adjustment of doses in therapy.